The present invention relates to modified enzymes, a skin care composition comprising said modified enzyme and ingredients known to be used in skin care composition, a skin care product comprising a skin care composition of the invention and the use of said modified enzyme for improving the stability and/or for reducing the sensitization potential of enzyme.
Since ancient time man has enjoyed taking baths and showers. This has not changed. For most people today bathing and showering are part of the daily rituals performed to maintain a good body hygiene and to obtain a pleasant scent. Certain people also regard a refreshing shower or bath in the morning as an important and necessary psychological experience without which they just cannot wake up.
A vast number of products for body care and maintenance of a good body hygiene, e.g. for cleansing and moisturising all parts of the body, are found on the consumer market. A few of these products comprise modified enzymes as an active ingredient.
Enzymes for Skin Care
The beneficial potential action of treating the skin with enzymes in the form of vegetables and fruits, such as cucumber, tomato, carrots, banana etc., have been known for a long period of time.
However, enzymes were not introduced into commercial skin care products before the 1970""ies, partly due to a limited knowledge about enzymes but also because enzymes were considered to have an unsatisfactory stability and also some disadvantageous properties in skin care products. For instance, cellulases were found to change the viscosity of lotions and creams containing carboxymethylcellulose; lipases resulted in changes in creams containing fatty acids esters; proteases were found to breakdown protein ingredients and to cause loss in viscosity.
Furthermore, also the high costs of enzymes at that time inhibited the application of enzymes in such personal care products.
The Human Skin
The human skin is composed of several layers. The top layer, the Epidermis, contains the fibrous protein keratin and functions as a sort of protective cover from the environment. The outer layer of the Epidermis is formed from organised cell death from the granular layer which lies underneath. In the granular layer numerous enzymes are released which convert the dead cell material to keratin.
The Corium (dermis) is connected to the Epidermis by way of the basal membrane and links the skin to the rest of the body through the circulatory system. The Corium is equipped with blood vessels, nerve fibres and lymphatic vessels and comprises a fibrous network of mainly collagen fibres with a limited amounts of elastin and reticulin fibres.
Modified Enzymes for Personal Care Products
As mentioned above some enzymes have an unsatisfactory stability and may under certain circumstancesxe2x80x94dependent on the way of contactxe2x80x94cause an immune response, typically an IgG and/or IgE response.
It is today generally recognised that the stability of polypeptides are improved and the immune response are reduced when polypeptides, such as enzymes, are coupled to polymeric molecules.
Techniques for conjugating polymeric molecules to polypeptides are well known in the art.
One of the first suitable commercially techniques was described back in the early 1970""ies (U.S. Pat. No. 4,179,337). Said patent concerns non-immunogenic polypeptides, such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene glycol. At least 15% of polypeptides"" physiological activity is maintained.
GB patent no. 1,183,257 (Crook et al.) describes chemistry for conjugation of enzymes to polysaccharides via a triazine ring.
Further, techniques for maintaining of the enzymatic activities of enzyme-polymer conjugates are also known in the art.
WO 93/15189 (Veronese et al.) concerns a method for maintaining the activity in polyethylene glycol-modified proteolytic enzymes by linking the proteolytic enzyme to a macromolecularized inhibitor. The conjugates are intended for medical applications.
It has been found that the attachment of polypeptides to polymeric molecules in general has the effect of reducing the activity of the polypeptide or interfering with the interaction between the polypeptide and its substrate. EP 183 503 (Beecham Group PLC) discloses a development of the above concept by providing conjugates comprising pharmaceutically useful proteins linked to at least one water-soluble polymer by means of a reversible linking group.
EP 471,125 (Kanebo) discloses skin care products comprising a parent protease (the Bacillus protease Esperase(copyright)) coupled to polysaccharides through a triazine ring to improve the thermal and preservation stability. The coupling technique used is described in the above mentioned GB patent no. 1,183,257 (Crook et al.).
JP 3083908 describes a skin cosmetic material contains a transglutaminase from guinea pig liver modified with one or more water-soluble substance such as PEG, starch, cellulose etc. The modification is performed by activating the polymeric molecules and coupling them to the enzyme. The composition is claimed to be mild to the skin.
Short Summary of the General Knowledge Based on Prior Art
Techniques for coupling one or more polymeric molecules to a polypeptide molecule are known in the art. Further, it is known that such modified enzyme-polymer conjugates have a reduced immune response and have an improved stability.
It is the object of the present invention to provide improved modified enzyme conjugates suitable for use in skin care products.
The present inventors have found that when using modified enzyme with an activity suitable for skin care certain claims must be imposed on the enzyme and polymeric molecule to obtain improved stability and a reduced sensitisation potential while still having a substantial residual enzymatic activity maintained.
The inventors found that the number and weight of the polymeric molecules coupled to the surface of the enzyme must be balanced with the weight and/or surface area of the enzyme. Further, the position of coupling the polymeric molecules are also of importance.
In the first aspect the invention relates to a modified enzyme having from 4 to 70 polymeric molecules, with a molecule weight from 1 to 35 kDa, coupled covalently to the surface of parent enzymes having a molecule weight from 15 to 100 kDa.
In a case of the parent enzyme has a molecule weight from 15 to 35 kDa from 4 to 20 polymeric are coupled covalently should be coupled to the surface of the enzyme.
If the molecule weight of the parent enzyme is in the range from 35 to 60 kDa from 7 to 40, preferably 10 to 30 polymeric molecules are coupled to the surface of said parent enzyme.
Likewise, if the parent enzyme has a molecule weight from 60 to 80 kDa from 10 to 50, preferably 13 to 40 polymeric molecules are coupled to the surface of said parent enzyme.
From 15 to 70, preferably 18 to 60 polymeric molecules are coupled to the surface of parent enzymes having a molecule weight from 80 to 100 kDa.
Normally polymeric molecules are coupled to the amino groups (xe2x80x94NH2) on the enzyme""s surface and the N-terminal amino group. However, polymeric molecules may also be coupled to the carboxylic acid groups (xe2x80x94COOH) of amino acids in the enzyme chain positioned on the surface.
Preferred attachment groups are Lysine residues and the amino groups at the N-terminal.
Carboxylic acid attachment groups may be the carboxylic acid group of Aspartate or Glutamate and the C-terminal COOH-group.
The number of xe2x80x9cattachment groupsxe2x80x9d in the present application also includes the number of the amino groups of Lysine residue in the polypeptide chain plus the N-terminal amino group.
The parent enzyme of the invention may be a hydrolase, including proteases, in particular subtilisins, or lipase, or an Oxidoreductase, including laccases and Superoxide dismutase.
In the second aspect the invention relates to skin care composition comprising a modified enzyme of the invention further ingredients being used in skin care products.
In the third aspect the invention relates to skin care product comprising a skin care composition of the invention.
The skin care product of the invention has improved stability and reduced sensitisation potential in comparison to corresponding skin care products (with parent enzymes).
The term xe2x80x9creduced sensitisation potentialxe2x80x9d means in the context of the present invention xe2x80x9creduced allergenicityxe2x80x9d which means that the amount of produced IgE (in humans, and molecules with comparable effects in specific animals), which can lead to an allergic state, is decreased when inhaling a modified enzyme of the invention in comparison to the corresponding parent enzymes.
In the context of the present invention xe2x80x9cskin care productsxe2x80x9d cover all personal care products used for cleansing, care and/or beautification of the skin of the body and further other products, such as hair care products, which during use might come in contact with the skin or respiratory system. Also corresponding products for animals are contemplated according to the present invention.
Specific examples of skin care products contemplated according to the present invention are soap, cosmetics, skin creams, skin gels, skin milk, skin lotion, cleansing cream, cleansing lotion, cleansing milk, cold cream, cream soap, makeup base, milky lotion, pack, calamine lotion, T zone essence, hand cream, essence powder, whitening powder, powder soap, cake soap, transparent soap, lip cream, lipstick, nourishing essence, creamy foundation, face powder, powder eye-shadow, powder foundation, nail polish remover, hair tonic, hair liquid, hair cream, hair gel, hair treatment, hair setting preparations, hair dyes, hair colorants, scalp treatment, shampoo, balsam, hair rinse, hair spray sun oil, sun screen, shaving foam and gel, shaving cream, baby oil, acne care products, antiperspirants, insect repellents, deodorants etc.
Assessment of Allergenicity
Assessment of allergenicity may be made by inhalation tests, comparing the effect of intratracheally (into the trachea) administrated parent enzymes with the corresponding modified enzymes according to the invention.
A number of in vivo animal models exist for assessment of the allegenicity of enzymes. Some of these models give a suitable basis for hazard assessment in man. Suitable models include a guinea pig model and a mouse model. These models seek to identify respiratory allergens as a function of elicitation reactions induced in previously sensitised animals. According to these models the alleged allergens are introduced intratracheally into the animals.
A suitable strain of guinea pigs, the Dunkin Hartley strain, do not as humans, produce IgE antibodies in connection with the allergic response. However, they produce another type of antibody the IgG1A and IgG1B (see e.g. Prentxc3x8, ATLA, 19, p. 8-14, 1991), which are responsible for their allergenic response to inhaled polypeptides including enzymes. Therefore, when using the Dunkin Hartley animal model, the relative amount of IgG1A and IgG1B is a measure of the allergenicity level.
A rat strain suitable for intratracheal exposure to polypeptides and enzymes is the Brown Norway strain. Brown Norway rats produce IgE as the allergic response.
The BALB/C mice strain is suitable for determining the IgE response caused by subsctaneous injection.
More details on assessing respiratory allergens in guinea pigs and mice is described by Kimber et al.,(1996), Fundamental and Applied Toxicology, 33, pp. 1-10.
Other animals such as rats, rabbits etc. may also be used for comparable studies.